Abstract P2-11-22: Expression of milk whey protein genes in human breast carcinoma cells isolated by LCM impacts prediction of clinical outcomes
Authors: James L. Wittliff, Michael W. Daniels
Journal: Cancer Research (2023)

Abstract Background: Milk whey protein consumption has been associated with breast cancer prevention, therapy and treatment tolerance, and other studies detected these proteins or their mRNAs in breast cancer models or tissues. Our objectives are to ascertain expression of certain whey protein genes in human breast carcinoma cells isolated by Laser Capture Microdissection (LCM) and to determine if gene expression is related to risk of recurrence and overall survival in lesions with various ER/PR status. Genes selected were: LALBA (alpha-lactalbumin), LPO (lactoperoxidase), LTF (lactotransferrin), PAEP (progestogen associated endometrial protein or glycodelin) and SSP1 (secreted phosphoprotein 1 or osteopontin). This retrospective investigation is unique in that highly quantified ER and PR protein levels and ESR1 and PGR relative expression from pure populations of carcinoma cells were employed to stratify cancers into ER/PR subcategories to assess relationships of expression of candidate genes with clinical outcomes. Methods: This investigation of primary breast carcinomas from 170 postmenopausal and 77 premenopausal patients used a matchless deidentified dataset of quantified estrogen receptor (ER) and progestin receptor (PR) protein results with clinical follow-up. ER/PR protein levels, expressed as fmol/mg cytosol protein, were quantified earlier from each breast carcinoma with FDA-approved kits, either Radioligand-Binding Assay (LBA, NEN/DuPont) and/or enzyme immunoassay (EIA, Abbott Labs). Patients were treated with the standard of care at the time of diagnosis. The PixCell IleTM (ThermoFisher, Arcturus) LCM instrument was used to procure only carcinoma cells from tissue sections of de-identified, frozen biopsies, using protocols we employed earlier. Total RNA of each fixed specimen was extracted, purified and amplified to obtain relative expression levels of approximately 22,000 genes by microarray, providing an assessment of mRNA in carcinoma cells only. Relationships of relative gene expression, quantified ER/PR and other features of primary breast cancers were analyzed with clinical results using R software v4.2.0 (Vigorous Calisthenics) by Univariable Cox Regression, Kaplan Meier plots and various tests (i.e., Wilcoxon, Log Ranked, Spearman and/or Chi Square tests). Bidirectional elimination stepwise regression was also used to derive best predictors of patient outcomes. Results: Violin plots of expression distribution of each milk protein gene of 247 biopsies regardless of patient menopausal status revealed that LALBA and SSP1 were highly elevated (p &amp;lt; 0.001) in ER+ (n = 146) or PR+ (n = 146) breast cancer cells while PAEP was quite diminished (p &amp;lt; 0.001). No difference was observed in LPO expression in ER+ or PR+ cells and LTF was only elevated slightly (p &amp;lt; 0.05) in ER+ cells. Regardless of ER/PR status of breast cancer, LTF, LALBA, SSP1, AR and ESR1 gene expression levels were increased significantly in postmenopausal versus premenopausal patients while PAEP was decreased (p &amp;lt; 0.001). No difference was observed in LPO or PGR expression. Correlation matrices examined gene to gene and receptor protein to gene relationships. Using Kaplan Meier analyses of each gene alone with Log Ranked Tests of outcomes, only SSP1 expression was associated with clinical outcome, i.e., overexpression was associated with lower PFS and OS (p = &amp;lt; 0.05). Conclusions: Application of bidirectional elimination stepwise regression of gene expression of the five milk proteins collectively with ER/PR protein status of each carcinoma revealed that LALBA gene expression enhanced prediction of PFS in ER+ cancer while PAEP expression was associated with improved prediction of OS in ER+ carcinomas. We report that expression of LALBA and PAEP genes, largely associated with milk production in normal breast tissue, are also expressed in certain breast carcinomas and influence prediction of patient survival. Citation Format: James L. Wittliff, Michael W. Daniels. Expression of milk whey protein genes in human breast carcinoma cells isolated by LCM impacts prediction of clinical outcomes [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-11-22.